Although disulfide bonds are critical to the structure of many secreted proteins, and to the regulation of a range of biochemical processes, their biosynthesis in multicellular organisms remains surprisingly cryptic. While oxidative protein folding has been regarded almost exclusively from an intracellular perspective, disulfide bond generation, isomerization and reduction also occur at the extracellular surface of mammalian cells. This application deals with an extracellularly-active FAD-dependent sulfhydryl oxidase (human Quiescin-sulfhydryl oxidase 1; QSOX1) that facilely introduces disulfide bonds directly into unfolded reduced proteins. QSOX1 is highly up-regulated in a number of human cancers (most notably of breast, pancreas and prostate) where it promotes adhesion and invasion of transformed cells. Members of the protein disulfide isomerase (PDI) family are also active exofacially where they support viral fusion, platelet aggregation, and the invasive phenotype of a range of cancer cells. The present work is directed towards understanding the activities of QSOX1 and PDI in key exofacial/extracellular thiol/disulfide (SH/SS) transformations. The first of three specific aims proposes the development of sensitive new fluorescence microscopy tools to evaluate surface SH/SS balance in a number of mammalian cell types. This aim combines two-color ratiometric imaging using both fixed and live cells and employs conventional and super-resolution fluorescence microscopies. The second aim explores the effects of knockdown of QSOX1 levels in human fibroblast cells on the SH/SS balance from the plasma membrane surface into the extracellular matrix. A comprehensive evaluation of the physiological substrates of QSOX1 in the extracellular space will inform the roles of the oxidase in adhesion and invasion. The third aim addresses the exofacial roles of PDI using a combination of redox imaging and the proteomic identification of isomerase substrates. New sensitive assays will be developed to evaluate the effectiveness of mono- and multifunctional arsenical and nitrostyrene derivatives as inhibitors of exofacial PDI. Overall, these three aims will contribute to a better understanding of the redox-enzymology of SH/SS transformations that play critical roles in mammalian cell behavior.